In this experiment, the peanut protein isolate (PPI), soybean protein isolate (SPI), rice bran protein isolate (RBPI), and whey protein isolate (WPI) were modified by linking chlorogenic acid covalently and linking dextran by Maillard reaction to prepare protein-chlorogenic acid-dextran (PCD) conjugates. As for structures, conformational changes of conjugates were determined by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis (SDS-PAGE), Fourier transform infrared (FT-IR), and fluorescence measurements. The molecular weights of PCD conjugates became larger, the structure became disorder, and the amino acid residues inside the protein were exposed to the polar environment when compared to protein-chlorogenic acid (PC) and native proteins (NPs). As for properties, the interfacial tension reduced and antioxidant activity of PCD conjugates enhanced in varying degrees. Based on this, PCD conjugates were used as emulsifiers in order to investigate the properties of nanoemulsions and compared with PC conjugates and NPs. The mean droplet diameters (MDD) results showed that the nanoemulsions that were stabilized by PCD conjugates had the smallest particle sizes and exhibited uniformly dispersed spherical shapes. The storage and oxidative stabilities of PCD conjugates were also significantly improved. In comparison, nanoemulsion that was stabilized by PPI-chlorogenic acid-dextran conjugate had the smallest particle size and optimal stability among four protein stabilized nanoemulsions. Nowadays, the nanoemulsion-delivery system has received widespread attention on encapsulating, protecting, and releasing lipophilic active ingredients, which was mainly due to their high stability and superior protective effects on carrier nutrients when compared to conventional emulsions . TherefoSome certain processing and storage conditions could alter the protein molecular conformation, aggregation state, and functional properties . The widThe protein-polysaccharide conjugates that were produced by Maillard reaction have attracted attention due to their increased functional properties, such as antioxidant activity, emulsifying properties, and thermal stability ,13. The In summary, the addition of polyphenols and polysaccharides both have effects on the protein structure and they could improve the protein emulsifying and other functional properties. Therefore, it was necessary to investigate the influence of protein-polyphenol-polysaccharide conjugates on the capability of emulsions preparation. Presently, it had been studied that lactoferrin-chlorogenic-dextran conjugate could improve the stability and bioaccessibility of β-carotene nanoemulsions as emulsifiers . HoweverDefatted peanut powder and rice bran powder were purchased from Changshou Food Co. . Low temperature defatted soybean powder was purchased from Harbin High-Tech Co. . Whey protein isolate was purchased from Jiangda Biotechnology Co. . Corn oil was purchased from a local grocery. Chlorogenic acid was purchased from Solarbio Co. . Dextran powder was purchased from Sinopharm Chemical Reagent Co. . 1,1-Diphenyl-2-picrylhydrazyl radical 2,2-Diphenyl-1-hydrazyl (DPPH), and 2,2′-amino-di(2-ethyl-benzothiazoline sulphonic acid-6)ammonium salt (ABTS) solid powders were purchased from Biotopped Co. . Nile red/blue were purchased from Sigma–Aldrich . Butylated hydroxytoluene (BHT) was purchased from Merck Co. . Trichloroacetic acid (TCA) was from Samchun Pure Chemicals Co. . Analytical-grade reagents were used, unless otherwise stated.The isolated proteins of peanut (PPI), soybean (SPI), and rice bran (RBPI) were extracted based on previous research ,17,18. IThe PC conjugates were prepared according to the method of Liu et al.  with sliv/v). The solution was adjusted to pH 7.0 and lyophilized. The obtained powder was incubated for 24 h at 60 °C, and the required 79% relative humidity (RH) was provided by saturated KBr solution.The PCD conjugates were prepared according to the method of Liu et al. , with slSDS-PAGE gel electrophoresis analysis was performed according to the Laemmli method . The NP,−1 resolution from 4000 cm−1 to 400 cm−1 at 25 °C. Peakfit Version 4.12  was used to perform baseline correction, deconvolution, and second derivative on the spectrum of the amide I band (1700–1600 cm−1). Gaussian peak shape was used to fit the number and position of sub-peaks and calculate the peak area. The corresponding relationship between each sub-peak and secondary structure were: 1610–1638 cm−1 and 1680–1690 cm−1 corresponding to β-sheet; 1640–1648 cm−1 corresponded to random curl; 1650–1660 cm−1 corresponded to α- helix; and, 1660–1680 cm−1 and 1690–1700 cm−1 corresponded to β-turns [The NP, PC, and PCD freeze-dried samples were analyzed by Fourier transform infrared (FT-IR) spectroscopy using a Bruker Vertex 70 FT-IR spectrometer . The FT-IR spectra were recorded with 64 scans at 4 cmThe fluorescence spectra of the NP, PC, and PCD samples were determined using a F-4500 Fluor photometer  according to Bonomi’s research with modifications . The samThe interfacial tension of the NP, PC, and PCD samples were determined using a Du Nouy ring tensiometer  at 25 °C. The loop was cleaned with acetone and then dried in a blue flame before each measurement. Repeat the experiment until the constant reading was obtained.A represented the absorbance of the sample and DPPH-ethanol mixed solution, iA represented the absorbance of the sample and the 95% ethanol solution, jA represented the absorbance of DPPH ethanol solution.1 mL of the NP, PC, and PCD samples were taken and added 4 mL of 0.1 M DPPH-ethanol solution, reacted in the dark for 30 min. at room temperature. The absorbance was measured at 517 nm (with a 95% ethanol solution as a reference). The calculated DPPH free radical scavenging rates according to the following Formula (1):+): dissolve 0.0384 g of ABTS solid powder in phosphate buffer solution , and then diluted to 10 mL with buffer solution to prepare the ABTS solution. Accurately weighed 0.0066 g of potassium persulfate and added to the solution to make the final concentration to 2.45 M, and stored for 16 h in the dark. The ABTS radical cation was diluted with a phosphate buffer solution to make the absorbance at 734 nm of 0.7 ± 0.02.The preparation of ABTS radical cation , and shaken for 30 s, then avoided light and reacted for 5 min. After reaction, measured the absorbance at 734 nm. The phosphate buffer solution was used as a blank control. The calculated ABTS free radical scavenging rates according to the following Formula (2):w/v) potassium ferricyanide solution. The reaction was carried out in a water bath at 50 °C for 20 min., then 2.5 mL of 10% (w/v) trichloroacetic acid was added and centrifuged at 5000 rpm for 10 min. when cooled. 2.5 mL of the supernatant was taken, added 2.5 mL of distilled water, and 0.5 mL of 1% (w/v) ferric chloride solution. Subsequently, the absorbance at 700 nm was measured after shaking and standing still for 10 min.Take 1 mL of the NP, PC, and PCD samples (5 mg/mL), added 2.5 mL phosphate buffer (0.2 M pH 6.6) solution and 2.5 mL 1% (w/v), SPI (Soy protein isolate), SPI-CA, and SPI-CA-Dex , RBPI (Rice Bran protein isolate), RBPI-CA, and RBPI-CA-Dex , and WPI (Whey protein isolate), WPI-CA, and WPI-CA-Dex  dried powder, dissolved in 10 mM pH 7.0 phosphate buffer solution to make the corresponding concentrations of aqueous solution, stirred at room temperature overnight to ensure complete dissolution of the powder.In aqueous phase, based on our previous research, accurately weigh PPI (Peanut protein isolate), PPI-CA, and PPI-CA-Dex . Subsequently, the ultrasonic processor  was used with ultrasonic power at 500 W for 20 min. The reaction temperature was controlled at 25 °C by ice water bath in order to prevent the reaction temperature from being too high during the preparation.The mean droplet diameters (MDD) of the samples were measured using dynamic light scattering . The refractive indices of the oil phase and the aqueous phase were set to 1.47 and 1.33, respectively. The zeta-potential (ZP) of the nanoemulsions was measured while using an electrophoresis . The samples were diluted 100-fold with 10 mM phosphate buffer (pH 7.0) to avoid multiple light scattering effects.−1. A 40 mm acrylic parallel plate with a geometric gap of 500 μm was slowly placed on the emulsion sample for 30 s and shear rates measurements were applied at room temperature.The apparent viscosity of the nanoemulsion samples was determined via a rheometer  at shear rates of 0.01–100 sThe nanoemulsions were stored at 4 °C for four weeks. The physical stability of the nanoemulsions was characterized at time intervals of one week. The physical stability was evaluated by measuring the change of MDD of nanoemulsions during the storage .The measurement of TBARS  was determined according to the method that was described by Nejadmansouri et al. . The TBAThe nanoemulsion samples were heated in a boiling water bath for 10 min. and 30 min, cooled to room temperature, and then diluted 100 times with 10 mM pH 7.0 phosphate buffer solution to measure the MDD of the samples .The nanoemulsion samples were stored in a refrigerator at −20 °C for 20 h and then thawed in a water bath at 35 °C for 2 h. The MDD of the samples were measured by diluting 100 times with 10 mM pH 7.0 phosphate buffer solution .p < 0.05) with SPSS V20.0 . Origin V9.1 , PeakFit V4.12  and other software were used for data processing, spectrum analysis, and chart making.All of the experiments were conducted in triplicate. The results were expressed as mean ± standard deviation. Significance of difference between the means was identified through the Duncan’s multiple-range tests  after absorbing ultraviolet light due to the existence of certain amino acid residues in the protein structure  . Previoumaxλ) of PCD samples exhibited red shift as compared to respective PC samples. It indicated that the addition of dextran changed the micro-environment of tryptophan in the protein, making it more polar to the water phase [The fluorescence intensities of the PCD conjugates were further reduced when compared to the PC conjugates. In fact, this phenomenon was probably due to the shielding effect of the dextran addition . The maxer phase . Therefoer phase . Among tp < 0.05). This revealed that the surface activity of the protein was enhanced through grafting with chlorogenic acid. Polyphenol modification could change the structure of protein, as analyzed in the FT-IR results. Protein molecules were de-aggregated, and the structures were further expanded. The connection of hydrophilic groups could reduce the interfacial tension and, thus, formed a more stable nanoemulsion.There has been correlation between interfacial layer rheology of protein and nanoemulsion formation and stability . The meap < 0.05). Among four PCD conjugates, PPI-CA-Dex had the smallest interfacial tension (7.63 ± 0.15 mN/m), while the RBPI-CA-Dex had the largest interfacial tension (11.87 ± 0.15 mN/m). In fact, after the addition of dextran, the dextran could further crosslink with the PC conjugates, leading to the partial unfolding of the protein at the adsorption layer. Thus, dextran induced further unfolding of the protein, as analyzed in FT-IR result, allowing for it to be more efficiently adsorbed at the oil-water interface. Subsequently, thicker viscoelastic membranes were formed with the reduction of the interfacial tension [It could be seen from  tension .p < 0.05). The results showed that PCD conjugates had the best effects on improving the antioxidant activity of the protein. Previous research indicated that the Maillard reaction was an effective method for improving the antioxidant properties, including free radical scavenging, iron chelating activity, and iron reducing ability [In addition, it could also be seen from the  ability . On the  ability . On the From the above discussion of the structure and properties of the protein, it could be seen that the polyphenol and polysaccharide modification of the protein could both make the protein secondary structure more disordered and the protein molecules further expanded. Thus, the emulsification properties were speculated to be better, which was further supported by the reduced interfacial tension. In addition, the antioxidant activity of the protein was also advanced with the bonding of polyphenol and polysaccharide. Therefore, further research on modified proteins used as emulsifiers to stabilize nanoemulsions owning high antioxidant ability attracted our interests.p < 0.05). After the modification of the polysaccharide, the absolute values of ZP decreased; this was not beneficial to the dispersion of the droplets. However, large molecular weight polysaccharides increased the steric hindrance of the protein, so it could also effectively prevent the aggregation and flocculation of the droplets. Zhao obtained similar results in the formation of WPI-Dex conjugates’ nanoemulsion [The ZP of the nanoemulsions stabilized by PPI-CA, SPI-CA, RBPI-CA, and WPI-CA changed from −42.77 to −45.53 mV, −34.40 to −37.30 mV, −30.50 to −31.87 mV, and −33.00 to −33.93 mV, respectively, as can be seen in emulsion .Rheological behavior was one of the most important properties in food emulsions. Emulsion products with good texture and sensory often required careful control of emulsion rheology . Figure Four PCD conjugates stabilized nanoemulsions had lower viscosity than respective PC conjugates nanoemulsions. This might be due to the decrease in the interfacial tension of the PCD conjugates after the addition of polysaccharides. The MDD of the nanoemulsion droplets stabilized by PCD conjugates became smaller than PC ones, resulting in smaller flow resistance in the flow field, as shown in p < 0.05), as shown in p < 0.05). However, the MDD changes of PPI and PPI-CA stabilized nanoemulsions were not significant (p > 0.05). This showed that PPI itself had strong storage stability as emulsifier. The WPI and WPI-CA stabilized nanoemulsions both increased particle size on the third week of storage, showing that the influence of chlorogenic acid on the stability of WPI-emulsified nanoemulsions was small.The MDD of nanoemulsions stabilized by SPI and RBPI increased significantly on the second week of storage (p > 0.05). Nanoemulsions stabilized by PCD conjugates as emulsifiers showed the best storage stability after the storage of four weeks. Firstly, the PCD conjugates had the lowest MDD in the initial stage, as shown in During the entire four-week storage, the MDD of nanoemulsions that were stabilized by four PCD conjugates all kept constant  could interact with thiobarbituric acid (TBA) to form a pink compound (TBARS). Its absorbance values can reflect the degree of oxidation of the nanoemulsion and could be detected at 532 nm. Therefore, TBARS was used to evaluate the oxidative stability of the emulsion. It can be seen from p > 0.05), as shown in p > 0.05). This might be because that PCD conjugates could form a dense and thick interfacial layer on the surface of the droplet to act as a space barrier as analysis in interfacial tensions, physically prevented the interaction of pro-oxidants and lipids, which in turn acted to inhibit the oxidation of nanoemulsions [Accordingly, the ability of PCD conjugates stabilized nanoemulsions was most improved. For four weeks storage, the TBARS values of the PCD conjugates stabilized nanoemulsions were all lower than those of the PC conjugates, as seen from mulsions . In addimulsions . The higp < 0.05). In general, as the heat treatment time was extended, the growth rate of droplet size accelerated and the aggregation between the droplets usually became more intense. The adsorbed protein partially expanded with the extension of the heat treatment time, further leading to the hydrophobic attraction to accelerate droplets aggregation [regation . Howeverregation ; (2) forp > 0.05 exhibited good resistance to droplet aggregation. The SPI-CA-Dex, WPI-CA-Dex and RBPI-CA-Dex stabilized nanoemulsions had smaller MDD increase after heat treatment for 30 min. than PC stabilized nanoemulsions. That was mainly because, when the protein grafted with dextran, the thermal denaturation temperature of the protein increased, which led to the improvement of the thermal stability of the PCD conjugates that were stabilized nanoemulsion.The MDD of PPI-CA-Dex stabilized nanoemulsions kept steady after heat treatment for 30 min. The MDD of the NP stabilized nanoemulsions changed greatly after the freeze-thaw treatment, as shown in p > 0.05). The spatial repulsion between droplets of PCD conjugates stabilized nanoemulsions was higher than that of PC ones, which was also the main reason to improve the freeze-thaw stability for PCD conjugates. Furthermore, from NP to PC conjugates to PCD conjugates, the adsorption effect at the interface got better because the interfacial tension became smaller, which gained more resistant to aggregation of the droplets, as can be seen in When compared with PC conjugates stabilized nanoemulsions, PCD conjugates stabilized nanoemulsions had the smallest increase in MDD. The MDD of PPI-CA-Dex, SPI -CA-Dex, WPI-CA-Dex, and RBPI-CA-Dex stabilized nanoemulsion increased by 0.34%, 7.20%, 5.79%, and 30.74%, respectively. Among them, the MDD of PPI-CA-Dex stabilized nanoemulsions did not change significantly after freezing and thawing (Herein, for the PCD conjugates, a combination of polyphenols and polysaccharides improved the emulsifying and antioxidant properties of native proteins. When compared with PC conjugates and NPs, the PCD conjugates had larger molecular weight according to the band that appeared at the upper end of the gel . After b


The most severe problem in bone regeneration is the defect in the interface. We prepared four types of implantation scaffolds of crosslinked gelatin (GE)/hydroxyapatite (HAp) to study the factors influencing interface interactions, they are film-crosslinked GE scaffold, gel-crosslinked GE scaffold, solid-crosslinked GE/HAp scaffold and gel-crosslinked GE/HAp scaffold. HAp could penetrate the entire GE matrix completely in four successive steps: physical preparation of a gel; chemical crosslinking; incubation in modified simulated body fluid (m-SBF) and freeze-drying. The penetrative nucleation and growth of HAp and the influencing factors in the GE matrix were investigated to ameliorate the interface interactions between organic and inorganic layers. During development of penetrative nucleation and growth, a tight connection was built between organic and inorganic layers, B-type carbonated HAp was formed after incubation with m-BSF, and the apatite content could be controlled. In summary, enhanced interface relies on not only the pre-seeded hydroxyapatite (HAp) as crystal nuclei but also the sufficient space for ions with high concentration to diffuse in. Functional substitutes for the repair and regeneration of bone have garnered increasing attention  is the best choice as “cement” because the human bone contains ~70% HAp  was purchased from Thermo Scientific . HAp was obtained from J&K Scientific . Glutaraldehyde (25% water solution) was purchased from Shantou Xilong Chemicals . Sodium carbonate and 1,4-dioxane were obtained from Beijing Chemical Works . Sodium chloride, potassium phosphate dibasic trihydrate, 4-(2-hydroxyethyl) piperazine-1-ethanesulfonic acid and calcium chloride (anhydrous) were purchased from Sigma–Aldrich . Sodium bicarbonate, potassium chloride, magnesium chloride hexahydrate, sodium hydroxide, and sodium sulfate were obtained from Sinopharm Chemical Reagents .Four types of scaffolds were fabricated using four procedures  as follov/v), 1,4-dioxane/water) for 6 h at 4°C. To maintain the microstructure and prevent the dissolution and swelling of the GE matrix, 1,4-dioxane/water (90/10) solvent was selected  was added to 10 mL of ultrapure water with vigorous magnetic stirring for 2 h at 50°C. The solution was coated in a 6-mm petri dish to form a cast film which was chemically crosslinked in a 1% glutaraldehyde solution  was immersed in 10 mL of ultrapure water with ultrasonic agitation for 30 min first. Then, GE (1 g) was added to this solution with vigorous magnetic stirring for 2 h at 50°C. Then, the mixture was poured into a 6-mm petri dish and cooled rapidly to 4°C to induce gelation. Afterwards, the scaffolds were chemically crosslinked in 1% glutaraldehyde solution for 6 h at 4°C. Gel-crosslinked GE scaffolds without seed crystal HAp was prepared by the same procedure.v/v), 1,4-dioxane/water) for 6 h at 4°C.Solid-crosslinked GE/HAp scaffolds were prepared. Briefly, HAp (0.05 g) was immersed in 10 mL of ultrapure water with ultrasonic agitation for 30 min. Then, GE (1 g) was added to this solution with vigorous magnetic stirring for 2 h at 50°C. Then, the mixture was poured into a 6-mm petri dish, frozen at −20°C for 24 h, transferred to a lyophilizer, and freeze-dried for 3 days. Afterwards, the scaffolds were chemically crosslinked in 1% glutaraldehyde solution (90/10 (All scaffolds were washed thrice with ultrapure water. This was followed by incubation in pure water (which was changed every 6 h) for 24 h to remove the remaining glutaraldehyde and 1,4-dioxane.2) and incubated in modified simulated body fluid (m-SBF) at 37°C, which was prepared as reported previously  employing a JSM-6700F  system.X-ray diffraction (XRD) analyses were undertaken using an X-ray diffractometer . Scaffold surfaces were scanned over a 2θ range from 5° to 60° at 2°/min.−1 at a resolution of 4 cm−1 and 32 scans. IR spectroscopy was done at 4,000–400 cm−1 at a resolution of 4 cm−1 and 32 scans.Fourier-transform infrared (FTIR) spectroscopy using a Tensor 27  system was used to characterize the structure of the formed apatite in scaffolds. For attenuated total reflection (ATR) analyses, spectra were recorded at 4,000–650 cmTo determine the degree of mineralization in scaffolds, thermogravimetric analysis (TGA) was done with a thermogravimetric analyzer . Experiments were undertaken at a heating rate of 5°C/min under air, and the temperature range was 50–700°C.2+ distribution can represent the apatite distribution. Hence, Ca2+ distribution was analyzed by EDS during SEM with an acceleration voltage of 15 kV.Energy dispersive X-ray spectroscopy (EDS) was carried out on an EDAX  system to evaluate apatite distribution in gel-crosslinked GE/HAp/apatite scaffolds. Ca2+ concentration of m-SBF on the first day of incubation (which denotes the mechanism of HAp nucleation) was measured using inductively coupled plasma-atomic emission spectroscopy (ICP-AES) employing an ICPE-9000  system.The variation in CaSince the implantation of artificial bone or bone cement is porous structure, gel GE scaffolds with or without HAp were crosslinked to simulate the natural bone structure and to find out the influence of HAp. To directly investigate the interaction of GE and HAp on the interface, a film of GE was crosslinked as comparison. Moreover, the mineralization of scaffold can be affected not only by the interface interaction of hybrid material and the crystal nuclei but also by the supplementary of crystal ions. The solid scaffold was crosslinked after freeze-drying to modify the ion diffusion channel.Different preparation designs lead to different mineralization process. For gel-crosslinked scaffolds, chemical crosslinking took place immediately after the physical gels had been formed. The large interspaces between networks of gels were retained, which provided many channels for the diffusion of water and ions. However, a freeze-drying procedure took place, and a porous structure was formed first for the solid-crosslinked GE/HAp scaffold. Simultaneously, the interspaces between GE molecular chains were compressed, and the pore walls became solid instead of loose. These differences in microstructure could result in different appearances of mineralization within scaffolds, which is essential for bone regeneration.For bone functional substitutes, structures that facilitate bone regeneration are necessary. A thin GE film led to a separation interface a. DirectTo investigate the apatite distribution and nucleation process in scaffolds, SEM was used to detect surface and cross-section morphologies. Gel-crosslinked GE/apatite, solid-crosslinked GE/HAp/apatite, and gel-crosslinked GE/HAp/apatite scaffolds are shown in Cross-section morphologies are shown in the insets of The large interspaces between gel networks in gel-crosslinked scaffolds provided channels for the diffusion of water and ions. Two requirements were needed to grow apatite into the GE matrix: (a) the matrix should be loose with large interspaces between macromolecular chains or networks to ensure free diffusion of water and ions; (b) there should be sites of strong nucleation within the matrix (these would be more competitive than carboxyl groups on the scaffold surface) to induce penetrative growth.After investigation about morphology and nucleation, crystal properties need to be confirmed. The surfaces of gel-crosslinked GE scaffolds, gel-crosslinked GE/HAp/apatite scaffolds, and rod-like HAp powder were assessed by XRD analyses . The XRD−1. The amide-II band (attributed to the bending vibration of N-H groups and stretching vibration of the C-N group) was observed at 1,530 cm−1. The amide-III band (illustrating the in-plane vibration of C-N and N-H groups of bound amide or vibrations of CH2 groups of glycine) was noticeable at 1,232 cm−1. In addition, the amide-A peak (representing NH– stretching coupled with hydrogen bonding) was observed at 3,291 cm−1, and the amide-B peak  was soft. This large difference in the modulus between the scaffolds incubated or not incubated in m-SBF  also ref2+ is shown in 2+ and the distribution of Ca2+ represented the distribution of c-HAp. The arrows above the images indicate the inner side of the scaffold. A large area of the scaffold was scanned by EDS to evaluate HAp distribution in the gel-crosslinked GE/HAp/apatite scaffold incubated in m-SBF for 21 days. The EDS diagram for Ca2+ and 2+ and 2+ and We hypothesized that the c-HAp distribution was related to the diffusion of Ca2+ in m-SBF  the surfaces, no HAp spheres formed in the inner regions of the scaffolds. Due to hindrance by networks of chains of GE molecules, the concentration of diffused Ca2+ and Based on the results obtained above, we put forward the following hypotheses for the crystallization mechanism in gel-crosslinked GE scaffold, solid-crosslinked GE/HAp scaffold and gel-crosslinked GE/HAp scaffold. As shown in 2+ and 2+ and 2+ binding to –COO− could be divided to two types: (i) close to the scaffold surfaces and (ii) in the inner regions of the scaffold. For (i), the concentration of Ca2+ and 2+ and 2+ and 2+ and in vitro. The supplementary of sufficient Ca2+ ions and the mechanical compliance of the scaffold with natural bone tissues need further investigations.GE/HAp scaffolds with HAp penetrating into the scaffold were prepared. The mechanisms of the nucleation and growth of HAp in the inner regions of the scaffolds were investigated. The scaffolds were fabricated successively by physical preparation of hydrogels, immediate chemical crosslinking, incubation in m-SBF, and lyophilization. c-HAp (B-type) was incorporated into the scaffolds, and c-HAp was very similar to the inorganic mineral found in bone. Fabrication using this method enabled the compressive modulus to be increased substantially, and the penetrative growth could be attributed to good interactions between HAp and GE macromolecules to solve the problem of interfacial forces. Also, the mechanisms of the nucleation and growth of HAp were clarified. That is, the loose structure matrix with larger interspaces between macromolecular chains, stronger inducive nucleation sites, and a high concentration of ions were needed. However, the unevenness of the distribution remained because the HAp crystal could grow on the surface of the matrix indeed easier than penetrate into the interior of the scaffolds. Therefore, a scaffold with a gradient of pore sizes  or a gradient of seed distribution could alleviate this problem. Furthermore, all the experiments in this study were carried out The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.critique of the manuscript for important intellectual content. All authors contributed to the final version of the manuscript and approved it for submission.SX and CH conceived and designed the study. ZZ, KL, and WZ carried out the experiments. ZZ, KL, WZ, and JG analyzed the data. KL and ZZ wrote the manuscript. YL offered a The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Rimicaris exoculata dominates the macrofauna inhabiting the active smokers of the deep-sea mid Atlantic ridge vent fields. We investigated the nature of the host mechanisms controlling the vital and highly specialized ectosymbiotic community confined into its cephalothoracic cavity. R. exoculata belongs to the Pleocyemata, crustacean brooding eggs, usually producing Type I crustins. Unexpectedly, a novel anti-Gram-positive type II crustin was molecularly identified in R. exoculata. Re-crustin is mainly produced by the appendages and the inner surfaces of the cephalothoracic cavity, embedding target epibionts. Symbiosis acquisition and regulating mechanisms are still poorly understood. Yet, symbiotic communities were identified at different steps of the life cycle such as brooding stage, juvenile recruitment and molt cycle, all of which may be crucial for symbiotic acquisition and control. Here, we show a spatio-temporal correlation between the production of Re-crustin and the main ectosymbiosis-related life-cycle events. Overall, our results highlight (i) a novel and unusual AMP sequence from an extremophile organism and (ii) the potential role of AMPs in the establishment of vital ectosymbiosis along the life cycle of deep-sea invertebrates.The symbiotic shrimp  Rimicaris exoculata  (Campylobacterota (previously Epsilonproteobacteria) , such as Mollicutes or Deferribacteres (The Pleocyemata shrimp ge (MAR) , 26. Thige (MAR) –29 and tge (MAR) , 30–34. ge (MAR) –37. Thisacteria)  is confiacteria) , 31, 32.bacteres –29 evokibacteres , 40. Thibacteres , 39. Howbacteres , 11.The immune system of crustaceans is based on cellular and humoral responses involving, among other substances, the production of AMPs . SeveralCrustins form a diverse and multigenic family of AMPs found in virtually all crustacean groups and in some hymenopteran insects . They arR. exoculata. Then, we investigated expression patterns in different host tissues and throughout its life cycle in order to identify possible correlations with the main symbiosis related events taking place at different life stages of this vent shrimp. These events include embryonic development  and Snake Pit , with the Research Vessel (R/V) Pourquoi pas? using the suction sampler of the remotely operated vehicle (ROV) Victor 6,000 and the human operated submersible Nautile during the oceanographic cruises BICOSE2014 (https://doi.org/10.17600/14000100) and BICOSE2 2018 (http://doi.org/10.17600/18000004) (8000004) . The iso8000004)  was used8000004) , and adu8000004) .FJ573157) and the oligo (dT)18 primer.The nucleotide sequence of the Re-crustin was obtained by RT-PCR using 2 μg of template cDNA, with the forward primer 5′- GACAAACACCTCCTCCTCCTCCA−3′ designed from the incomplete 5′ coding sequence of a crustin sequence available in GenBank (accessing number ® (IKA). RNA was extracted according to the manufacturer's instructions. The concentration of extracted RNA was estimated with the Qubit® 3.0 Fluorometer (ThermoFisher Scientific). RNA extracts were treated by RQ1 RNase-free DNase (Promega) and used for cDNA synthesis with the RevertAid M-MuLV RT kit (ThermoFisher Scientific) according to the manufacturer's protocol. Reaction mixtures for PCR amplifications contained 0.1 μM of each primer, 0.25 mM of each desoxynucleotide triphosphate, 5 × Go Taq G2 Flexi buffer (Promega), and 5 U of GoTaq G2 Flexi DNA polymerase (Promega). The PCR program involved an initial denaturation step at 95°C for 3 min, followed by 39 cycles of 95°C for 1.5 min, 55°C for 1.5 min, and 72°C for 1.5 min, with a final elongation step at 72°C for 5 min.Whole animals were ground in Trizol ReagentTM using the Ultra-Turrax T25® Gel and PCR Clean-up kit (Macherey-Nagel). Purified PCR products were cloned using the TOPO-TA kit . Clones were sequenced according to the Sanger method (http://www.geneious.com/). Prediction of signal peptide was performed with the SignalP 4.1 program . Total RNA extraction and RT were performed as described in the previous section. The primers used for the quantitative PCR were designed with the Primer3 Input software .Tissues from branchiostegite, scaphognathite, gills, abdomen, stomach, hepatopancreas and eggs dissected aboard as wells as whole adults and juveniles were ground in Trizol ReagentTM using FastPrep-24- crustin primers: forward: 5′-ACTGCTGTGAGAACGGGAAC-3′; reverse: 5′-AACATGTTTGAGGGGGTCCT-3′- Rpl8 primers: forward: 5′-GAAGCTCCCATCAGGTGCCAAGAA-3′; reverse 5′-TTGTTACCACCACCGTGAGGATGC-3′.® 480 system (Roche) using a hot start enzyme. RT-qPCR assays were submitted to an initial denaturation step of 10 min at 94°C followed by 40 cycles of denaturation at 94°C for 15 s, annealing at 59°C for 1 min and extension at 72°C for 30 s. Reference and target were amplified in separated wells. After amplifications, a melting curve analysis was performed in order to confirm the specificity of the PCR products. Re-crustin primers generated a single and discrete peak in the dissociation curve (data not shown). A negative control and a 5-fold dilution series protocol of pooled cDNAs were included in each run. The 5-fold dilution series were used to construct a relative standard curve to determine the PCR efficiencies and for further quantification analysis. In all experiments, all primer pairs gave amplification efficiencies of 90–100%. Each reaction was run in triplicates. Analysis of relative gene expression data was performed using the ΔΔCq method  was coupled to ovalbumin and used for the immunization procedure of two New Zealand White rabbits (Saprophyte Pathogen free) according to the protocol of Covalab™ (France).Total proteins were isolated from the samples used for RNA extraction according to the manufacturer's instructions . The white band (interphase) containing the proteins was washed with a solution of 0.3 M guanidine hydrochloride in 95% ethanol, and then resuspended in 9.5 M urea and 2% CHAPS. The protein concentration was determined by the Bradford method using BSA as a standard . Protein2). After transfer, the gel was stained by Coomassie Brilliant Blue R-250 (BioRad). The membrane was blocked for 1 h in PBS at 0.1 M containing 0.05% Tween 20 and 5% casein and was then probed with the rabbit polyclonal anti-Re-crustin antibody (1:300 dilution) in the blocking solution (PBS at 0.1 M with 5% w/v non-fat dry milk) overnight at 4°C. After three washes with PBS/0.05%-Tween 20, the membrane was incubated for 1 h in the blocking solution at room temperature with the peroxidase-conjugated anti-rabbit secondary antibody Abcam . A Clarity™ Western ECL Substrate (Bio Rad) was used for the chemoluminescence visualization of the immunolabeling with a Kodak Bio Max light film.The proteins of the SDS-PAGE gel were transferred to a nitrocellulose membrane 0.2 μm (BIO RAD) by semi-dry electro blotting , juveniles and adult tissues (thickness of 7 μm). Consecutive paraffin sections were made with a LEICA RM 2255 microtome. Immunocytochemistry and immunohistochemistry were performed with the rabbit anti-Re-crustin (1:400) and the FITC-conjugated anti-rabbit secondary antibody . Samples were examined using a confocal microscope (Zeiss LSM LSM780) and the Fluorescence microscope (Zeiss Axio Imager 2).Micrococcus luteus and one Gram-negative Vibrio diabolicus were chosen for being easily carriable and cultivable onboard a ship. M. luteus routinely used in laboratory, is found in soil, dust, water and air and V. diabolicus was isolated from deep sea hydrothermal vents  in 0.1 M PBS at 4°C. 10 μL of samples were incubated without (control) or with 0.5 μl of the anti-Re-crustin antibody (dilution 1:400) at 4°C for 20 min.M. luteus or alive V. diabolicus (1 × 105 Colony Forming Unit (CFU)/mL of LB agar). After an overnight incubation at 37°C, the activity was quantified by measuring the diameter of the bacterial growth inhibition.10 μL of each sample were spotted onto LB-agar (Luria-Bertani) plates containing alive Pourquoi pas? R/V during the BICOSE2 2018 cruise with freshly dissected tissues and twice back to the laboratory in Lille with tissues frozen during the same cruise.Experiments were performed in triplicate, once aboard the R. exoculata (GenBank accession number: MT102281). Only one sequence of crustin was identified from our molecular subcloning and sequencing. The complete cDNA sequence encodes a precursor of 190 amino acid residues, which includes a 15-residue signal peptide , the morotoge shrimp Pandalopsis japonica , the Japanese spiny lobster Panulirus japonicus  and the Chinese mitten crab Eriocheir sinensis . Within Type II crustins from penaeid shrimp (Dendrobranchiata), Re-crustin was 52–59% identical to Type IIa crustins and 44–53% identical to Type IIb crustins. On the other hand, the mature Re-crustin displayed 36–44% identity to Type I crustins from decapods from both Pleocyemata and Dendrobranchiata suborders. Less than 30% amino acid identity was observed between Re-crustin and Type III (single WAP domain-containing proteins or SWD) and Type IV crustins (double WAP domain-containing proteins or DWD). Western blot analysis from total protein extracts showed a band at the predicted molecular weight confirming (i) the specificity of the antibody designed to recognize the WAP domain of Re-crustin and (ii) the translation of the Re-crustin transcripts ., 47.5, 4Because the life of adults is punctuated by molt cycles, the same protocol was applied to “white” adults at the beginning of their molt cycle, where almost all epibiont have been eliminated, in comparison with “red” adults at the end of their molt cycle, highly colonized but where epibionts are encrusted in minerals impairing their activities  Figure . In bothR. exoculata.We open hypothesis for a novel biological role for gene-encoded antimicrobial host defense peptides (AMPs) in crustacean-microbe interactions. Our results revealed that the expression of a new member of the classic crustin AMP family (Re-crustin) is spatio-temporally correlated with the establishment of the ectosymbiotic microbial communities inhabiting the cephalothoracic cavity of the extremophile deep-sea shrimp R. exoculata expresses in its tissues a N-terminal glycine-rich crustin that belongs to the Sub-Type IIa. Indeed, the Type II comprises a distinct group of crustins usually found in penaeid shrimp (Dendrobranchiata suborder) that is subdivided into two Sub-Types: Type IIa (“Crustins”) and Type IIb (“Crustin-like”)  . Althougn-like”) , 60. On n-like”) .in vivo. This interspecific interaction appears as an important function of Re-crustin in R. exoculata. Our multiple approaches all demonstrate that Re-crustin is essentially produced by the mouthparts in the cephalothoracic cavity, which is hugely colonized by the ectosymbionts  showed antibacterial activity against both Gram-positive and Gram-negative bacteria  without killing them. A control of the bacterial growth via bacteriostatic activities of Re-crustin as observed in the endosymbiostasis of beetles cannot be excluded . Further investigations should be performed to determine whether the expression sites are the neurons themselves or the epithelial cells infiltrated into the nervous system as documented for the crustin named PET-15 in the spiny lobster . A multiRimicaris, we explored the spatio-temporal correlations between the levels of production of Re-crustin and the ectosymbiotic acquisition/loss/re-colonization events that punctuate the life cycle of the hydrothermal shrimp  together with the loss of the attached ectosymbiotic community, notably on scaphognathites and branchiostegites in our case. Interestingly, the production of Re-crustin by the scaphognathites and branchiostegites reaches a peak when the re-colonization occurs, suggesting a role of Re-crustin in the control of the symbiosis acquisition and presumably in the selection and success of the appropriate bacteria from the habitat. Surprisingly, the protein load is increased in the pre ecdysis stage while the mRNA expression of Re-crustin is not. In addition to the technical explanation (see results), a neosynthesis might also occur in between the two stages  and should be investigated. An increased gene expression starting after the post ecdysis would lead to an accumulation along the molt cycle of the protein (embedding the symbionts that have colonized the gill chamber) which quantity would reach a maximal at the pre-ecdysis stage whereas the mRNA transcription would have already stopped. Since Rimicaris forms large and dense colonies constituted by individuals desynchronized in their molting and development, the symbiotic bacteria remain always available within the population, such as on shed exuvia, thus allowing the horizontal transfer of the symbionts. Because Re-crustin accumulates on the surface of both the symbionts and the cuticle of the appendages, the molecule may also serve as a communication pathway (such as a chemoattractant effect see before) in between the host and the bacteria thus favoring the recruitment and the horizontal transfer of the free-living symbionts.Like most crustaceans and contrary to metabole insects, R. exoculata molt cycle could be compared to the concept of symbiotic trade-off retrieved in insects (During the extremely short molt cycle (10 days), the symbiont growth is accompanied by a progressive mineral accumulation, caused in part by the symbiont activities , 70. In  insects  whereby R. exoculata enlarged cephalothoracic cavity offers a mechanic but also, an immune protective and growth environment for the symbionts against the harsh hydrothermal vent conditions and their microbial competitors while the symbionts provide nutrients via a transcuticular transfer (via the production of Re-crustin would have to be investigated to decipher the correlated production of the AMP with the symbiotic colonization and the transition stages of the host.Our results overall highlight a novel AMP sequence from an extremophile organism and suggest the role of AMPs in the establishment of vital ectosymbioses that take place along the life cycle of marine organisms. transfer  and maybMT102281.The datasets generated for this study can be found in the Re crustin, GenBank accession number: AT, M-AC-B, and FP designed the research. SL, CB-W, VC-G, LD, and PM developed reagents and performed experiments. SL, CB-W, RR, VC-H, M-AC-B, and AT analyzed the data. SL, CB-W, RR, VC-H, PM, FP, M-AC-B, and AT wrote the manuscript. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Japalura splendida  is determined and analyzed in this study. It contains 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and one non-coding regions. All the genes in J. splendida are distributed on the H-strand, except for the ND6 gene and seven tRNA genes which are encoded on the L-strand. The phylogenetic tree suggests that J. splendida and Japalura flaviceps formed a sister group and reveals the order , , Pseudocalotes microlepis))), Calotes versicolor) with substantial support for the monophyly.The complete mitogenome of  Japalura splendida  belongs to the family Agamidae, suborder Lacetilia and Oder Squamata. This lizard occurs only in China . In the last two decades, numerous mitogenomes of vertebrates have been sequenced and improved to obtain a reliable phylogeny , China in 2018 and muscle tissue was taken and preserved in 100% ethanol. Total genomic DNA was extracted from the tissue and stored in −80 °C ultra-low temperature freezer using Ezup-pillar Genomic DNA Extraction Kit . A DNA sample was shipped to Personal Biotechnology Co, Ltd  for library construction and sequencing by next-generation sequencing (NGS) methods  in length and contains 37 genes  and 1 control region (D-loop). The overall nucleotide compositions of this mitogenome are A (34.88%), T (23.74%), C (28.02%), and G (13.35%), respectively with 58.63% A + T content. In this mitogenome, ND6 gene and seven tRNA genes  were encoded in the L-strand, while the rest of genes were encoded in the H-strand. The tRNAs varied from 53 to 74 bp in size, and the lengths of 12S rRNA and 16S rRNA were 843 and 1461 bp, respectively. In addition, the control region was 1482 bp long and is located between the tRNA-Pro and tRNA-Phe genes. The gene arrangement pattern and transcription directions were identical to previous studies on Japalura flaviceps  methods with the concatenated nucleotide sequences of 13 protein-coding genes ), , Pseudocalotes microlepis))), Calotes versicolor) with high bootstrap support values (Twenty-one mitogenome sequences including 20 Agamidae and one t values . The phyt values .


SARC-F is a quick questionnaire recommended as a screening tool for sarcopenia. The aim of the study was to translate, adapt, and validate the Polish version of the SARC-F for community-dwelling older adults in Poland.We included 160 Polish volunteers aged ≥ 60 years (44% of men). The Polish version of SARC-F was adapted following standardized forward-backward translation procedure. SARC-F was validated against the six sets of diagnostic criteria as the reference standards .SARC-F score ≥ 4 points was observed in 18.8% of the study population. Cronbach’s alpha was 0.70. The sensitivity of SARC-F varied from 33.3% to 50.0% depending on the diagnostics criteria used, while the specificity was about 85%. Positive predictive value (PPV) was low (about 30%) for five out of six sets of the diagnostic criteria used , while the negative predictive value (NPV) was generally high (>88%). The area under the ROC curves (AUC) was 0.652–0.728. SARC-F had the largest AUC against FNIH criteria (0.728), indicating a moderate diagnostic accuracy. Similar results were found for EWGSOP2 and IWGS criteria. The AUC values were below 0.7 for AWGS, SCWD, and EWGSOP1 criteria.Based on the results, the Polish version of SARC-F shows excellent reliability and good internal consistency. High specificity and high NPV make SARC-F a useful tool to rule-out sarcopenia with high accuracy in community-dwelling older adults, independently of the diagnostic criteria used. Sarcopenia, a progressive and generalized loss of muscle strength, function and mass, is a severe condition threatening healthy aging . In 2013Diagnostics of sarcopenia is complicated, as the assessment of muscle mass and function requires specialized equipment and is time-consuming. Such measurements are an inherent part of all currently available sets of diagnostic criteria for sarcopenia, including European Working Group on Sarcopenia in Older People EWGSOP1) , the Ext , the ExTo extend the SARC-F questionnaire’s availability, Special Interest Group on Sarcopenia of European Union Geriatric Medicine Society (EuGMS) included language translation and validation of SARC-F among their priorities in 2017 . Such anThe reliability and validity of the SARC-F questionnaire were assessed in a cross-sectional study conducted between March 2019 and February 2020 in community-dwelling volunteers living in a city of Poznan, one of the largest cities in Poland. The inclusion criteria were age (60 years and older), preserved cognitive function [defined as Abbreviated Mental Test Score (AMTS) ≥ 7 points], ability to maintain standing position (necessary for assessment of body height and composition and Appendicular Lean Mass (ALM) estimation), and the ability to walk a 4-meter distance (to assess the gait speed). Subjects in whom the analysis of body composition with the bioimpedance method (BIA) was impossible because of implanted artificial pacemaker, metal implants, or oedemas were excluded. One hundred seventy persons volunteered for the study. Ten of them were excluded for the following reasons: cognitive impairment (n = 5), having a pacemaker (n = 2), physical disability preventing a 4-m usual walking speed test (n = 3).Each subject gave written informed consent before they participated in the study. The study was conducted in accordance with the Declaration of Helsinki, and the protocol was approved by the Poznan University of Medical Sciences, Poland .Face-to-face interviews were conducted to collect sociodemographic  and clinical (self-reported comorbidity and number of drugs taken regularly) data. Anthropometric measurements were taken . Nutritional status was screened with Mini Nutritional Assessment (full MNA), independence in basic activities of daily living (ADL) was assessed with the Katz scale, and independence in instrumental activities of daily living (IADL) with the Lawton Scale. The cognitive performance was assessed with the Abbreviated Mental Test Score (AMTS). All the tools used in the study have been described in detail in our previous publication .The SARC-F questionnaire comprises five domains: 1) strength, 2) assistance in walking, 3) rising from a chair, 4) climbing stairs, and 5) falls. Each domain is scored 0–2, and total score ≥ 4 indicates the risk of sarcopenia  was calculated in all subjects. Height was measured with a mobile stadiometer . The cut-off points used for the ALM index are shown in The muscle mass was measured with the BIA method . The Appendicular Lean Mass (ALM) index [defined as the sum of lean mass of upper and lower limbs (kg) divided by the squared height  and using The Chair Stand Test (CST). The handgrip strength test was performed in a sitting position, with arms bent to 90 degrees in the elbow and shoulder joint. Measurements for the left and right arms were repeated twice, and the mean value was calculated to give the final score. The results were expressed in kilograms (kg). According to the EWGSOP2 algorithm , we addiThe 4-m usual gait speed test (UGS) was used to assess physical performance. Subjects were allowed to use walking aids  during the test, if necessary. The time necessary to complete the test was measured with a stopwatch. The results were expressed in meters per second. The cut-off points for the UGS test are presented in SARC-F was evaluated used the following procedures: (1) the reliability was obtained by the Cronbach alpha and the correlation of each item in the questionnaire with the scale’s total score; (2) the temporal consistency was evaluated by test-retest ; (3) the criterion validity was assessed through the calculation of the sensitivity, specificity, and positive and negative predictive values of the SARC-F concerning sarcopenia defined according to the EWGSOP1, EWGSOP2, AWGS, IWGS, FNIH, and SCWD criteria; and (4) the associations between SARC-F and physical measurements, such as handgrip strength, CST, UGS, ALM, ALM/BMI, ALM index, and BMI. The SARC-F scale was also validated against other measurements related to sarcopenia, such as (1) age, (2) the Katz Scale to assess basic activities of daily living (ADL), (3) the Lawton Scale to assess instrumental activities of daily living (IADL), (4) the Mini Nutritional Assessment (MNA) to measure the risk of malnutrition. The total score obtained in all the scales was considered.Statistical analysis was performed using the STATISTICA 12.0 package . Continuous data were presented as mean ± SD and compared using the Student’s t-test or the Cochran–Cox test, or Mann–Whitney test as appropriate. Categorical variables were expressed as numbers (percentage) and compared with the χ2 test with the Yates correction if applicable. Men and women results were analyzed separately. Reliability was assessed by internal consistency, inter-rater, and test-retest analyses. Internal consistency was tested using Cronbach’s alpha for all five items. Cronbach alpha value ≥0.70, indicating an acceptable level of internal consistency . The tesROC analyses for the SARC-F based on the six sets of international diagnostic criteria [i.e., reference standard: EWGSOP1 , EWGSOP2One hundred sixty persons aged ≥ 60 years  were included in the study. Clinical characteristics of the total study population and classified by sex are presented in More than one-third of the study participants was ≥ 75 yrs. Women were older than men . Almost half of the participants  were unmarried, including widows/widowers (n = 52), those who were divorced or separated from their spouses (n = 7), as well as those never married (n = 8). One-third of the study population was living alone . The majority of subjects had secondary education or higher.2). Almost one out of eight persons had low BMI  [Women were significantly shorter than men (156.8±6.0 vs. 172.1±6.4 cm) and had lower body mass (66.8±14.7 vs. 79.5±14.0 kg), but their BMI did not differ significantly (27.2±6.1 vs. 26.8±4.4 kg/mriteria) . The pre2; p<0.001). The SARC-F scores were higher in women as compared to men .Upper limb muscle strength was higher in men than in women . Lower limb strength assessed with the Chair Stand Test was similar in both sexes. However, the 4-m usual gait speed tended to be lower in men . Women had lower ALM  and ALM Index (6.4±1.0 vs 7.9±1.0 kg/mTwenty volunteers (10 women and 10 men) aged 65–91 years (mean 76.0±7.3 yrs) were independently examined by two researchers (a physician-geriatrician and a physiotherapist) in two different offices. The mean age was 77.4±7.5 yrs in women and 74.6±7.2 yrs in men (p>0.05). Inter-rater reliability was determined by ICC. There was an excellent agreement between the results obtained by the two researchers, as indicated by the ICC value 0.993, and a very strong positive correlation . The same 20 persons were re-examined 2–4 weeks later by one of the researchers (the physiotherapist) for the test-retest reliability check. The results were concordant in 75%. The intraclass correlation coefficient for test-retest was 0.9280 (p<0.001), indicating an excellent agreement.As shown in The translated and culturally adapted Polish version of the SARC-F questionnaire showed excellent inter-rater reliability and test-retest reliability, and acceptable internal consistency . The SARC-F internal consistency was previously assessed in some validation studies; most of them gave results similar to those obtained in our study. For example, Malmstrom et al. found a very high Cronbach’s alpha (0.76–0.81) for the original version of SARC-F . In 2019In line with the results of some previous studies, we have demonstrated a correlation between SARC-F and some parameters associated with sarcopenia, such as age, low gait speed, low upper and lower limb strength, and low ALM , 20, 24.The results of clinical validation of the Polish version of SARC-F against six sets of international diagnostic criteria for sarcopenia showed low sensitivity , but high specificity  and high negative predictive value . Therefore, the SARC-F questionnaire is a suitable tool for excluding patients without sarcopenia. A negative result of SARC-F makes the diagnosis of sarcopenia less probable, thus eliminating the need for costly diagnostics and the use of specialist equipment  or BIA for muscle mass assessment).We have analyzed the performance of the Polish version of SARC-F against all currently available sets of international diagnostic criteria for sarcopenia. A similar approach was adopted in some earlier SARC-F validation studies, assessing the psychometric properties of this questionnaire against at least three diagnostic algorithms , 21, 25.To the best of our knowledge, the lowest sensitivity of SARC-F against EWGSOP1 diagnostic criteria was found in a validation study conducted by Woo et al.  in a popOne limitation of our study is the use of the BIA method for muscle mass assessment instead of more precise magnetic resonance imaging (MRI), computed tomography (CT) or DEXA. While the evaluation of muscle mass based on whole body MRI is regarded as the gold standard, rarely it is used in clinical practice and research studies involving larger populations due to its high cost . RegionaA strong point of our SARC-F validation analysis is that we were the first to use all currently available sets of international diagnostic criteria for sarcopenia as a reference standard [there are six of them, developed independently by European Working Group on Sarcopenia in Older People 1 (EWGSOP1) , EuropeaThe Polish version of the SARC-F questionnaire shows excellent reliability and good internal consistency. High specificity and high negative predictive value make SARC-F a useful tool to rule-out sarcopenia with high accuracy in community-dwelling older adults, independently of the diagnostic criteria used. Screening with the SARC-F questionnaire will enable reducing unnecessary and costly measurements required by all currently available sarcopenia diagnostic criteria sets.S1 Table(DOCX)Click here for additional data file.


Cordyceps militaris, a well-known edible and medicinal mushroom, were studied. The distribution of hydrophobins in ascomycetes with different lifestyles showed that pathogenic fungi had significantly more hydrophobins than saprotrophic fungi, and class II members accounted for the majority. Phylogenetic analysis of hydrophobin proteins from the species of Cordyceps s.l. indicated that there was more variability among the class II members than class I. Only a few hydrophobin-encoding genes evolved by duplication in Cordyceps s.l., which was inconsistent with the important role of gene duplication in basidiomycetes. Different transcript patterns of four hydrophobin-encoding genes during the life cycle indicated the possible different functions for each. The transcripts of Cmhyd2, 3 and 4 can respond to light and were related with the photoreceptors. CmQHYD, with four hydrophobin II domains, was first found in C. militaris, and multi-domain hydrophobins were only distributed in the species of Cordycipitaceae and Clavicipitaceae. These results could be helpful for further function research of hydrophobins and could provide valuable information for the evolution of hydrophobins.Hydrophobins are a family of small secreted proteins found exclusively in fungi, and they play various roles in the life cycle. In the present study, genome wide analysis and transcript profiling of the hydrophobin family in  Hydrophobins are small surface-active proteins produced exclusively by filamentous fungi ,2. They According to the spacing between the conserved cysteine residues, distinct hydropathy patterns and physical properties, hydrophobins are traditionally divided into two classes (class I and class II) ,9. ClassSchizophyllum commune, which plays an essential role in the formation of aerial mycelium . In . In C. mophobins .C. militaris, and the β-hairpins of each subunits conducted a β-barrel  was observed C.Cordycipitaceae and Clavicipitaceae. Five species in each family had been found to contain multi-domain hydrophobin proteins, and each species had only one except B. bassiana (3) and T. hemipterigena (2) (T. hemipterigena (CEJ88606.1) had the most (seven) domains. All the multi-domain hydrophobins were acidic proteins, except that in P. chlamydosporia (RZR67753.1).A survey of multi-domain hydrophobin proteins in the published genome of fungi from GenBank revealed that they were only distributed in the fungi of gena (2) . These mgena (2) . There wC. militaris is considered as a model organism for the study of over 400 species of Cordyceps [C. militaris was studied. The distribution of hydrophobins in ascomycete fungi and the evolution of hydrophobins in Cordyceps s.l. were also analyzed. It was found that four hydrophobin genes had different responses to light irradiation and different relative transcript levels during the life cycle. The ascomycete fungi with a pathogenic lifestyle had significantly more hydrophobin-encoding genes than saprotrophic fungi. More variability among the class II members was observed than class I, and only a few paralogous proteins were evolved by duplication. Multi-domain hydrophobins were only distributed in the species of Cordycipitaceae and Clavicipitaceae. The results could be helpful for further function research of hydrophobins in C. militaris, and could provide valuable information for the evolution of hydrophobins.Hydrophobins are a family of small secreted proteins found exclusively in fungi, and they play various roles in the life cycle of fungi. ordyceps . In thisBjerkandera adusta and Phlebia brevispora had only six out of eight conserved residues, but the missing ones were the first and the sixth residues [Among the four hydrophobins, two were identified as class I members and the others were class II. According to the alignment of amino acid sequences, three owned conserved eight cysteine residues except that CmHYD2 lacked one cysteine in the two CC doublets, respectively . The homresidues . Based oresidues ,38,39. Tresidues . In addiresidues  as well Cmhyd1 and Cmqhyd presented on chromosomes VI and IV, respectively. The strength of linkage between two genes depended upon the distance between the genes on the chromosome [Cmhyd4 was separated by a long distance from Cmhyd3 and Cmhyd2. They are obviously unlinked. Whether Cmhyd3 and Cmhyd2 are linked together should be verified by the construction of linkage maps.The location of the genes encoding four hydrophobins and one quadr-hydrophobin on the chromosomes were firstly analyzed . Cmhyd1 romosome . Though Though there was only class I hydrophobin, a significant expansion of hydrophobin-encoding genes in basidiomycetes was reported (1–40 copies) . In the Cordyceps s.l. The amino acid sequences from C1 to C8 were aligned because of the poor amino acid sequence conservation of the hydrophobins. The result strongly supported the two major clades, class I and class II, with relatively high bootstrap values, which was consistent with the previous report [Purpureocillium lilacinum; PHH79106.1, PHH68338.1 and PHH65630.1 from O. australis; PHH60601.1 and PHH71624.1 from O. australis; CEJ93582.1 and CEJ93581.1 from Torrubiella hemipterigena. Most of these twins formed a terminal branch, or were connected by a single node, indicating that they were aroused by gene duplication. However, CmHYD3 and CmHYD4, and CmHYD1 and CmHYD2 were not produced by gene duplication. Both birth-and-death evolution [Phylogenetic analysis of hydrophobins from both basidiomycetes and ascomycetes ,8, a reps report  because volution  have beeC. militaris indicated there may be different functions. Cmhyd1, one of the class II members, showed a high expression compared with HY stage during the whole life cycle cultured on both insect and wheat media , CmQHYD, was found during the search for hydrophobins in the genome of ilitaris . The muled cfth1 ,44. Therlication .Cordycipitaceae and Clavicipitaceae  and disruption strains of photoreceptor Cmwc-1  and Cmv were maC. militaris genomes (Accession: SRA047932 and PRJNA323705) [C. militaris genomes by Basic Local Alignment Search Tool (BLAST) to ensure finding all hydrophobin members [Two sequenced A323705) ,46 were  members . The pre members . Hydroph members .https://swissmodel.expasy.org/) . Firstly, the sequence of hydrophobins was submitted to the SWISS-MODEL server to determine the optimal template for model building. Then, we ranked the top 10 matches according to their sequence identity and QMEAN4 scores. The optimal templates were selected to build alternative models for hydrophobins. The identical network of disulfide bridges was added with PyMOL software [Homology modeling of hydrophobins including CmHYD1–CmHYD4 and CmQHYD was constructed by the SWISS-MODEL server (software .−5), (2) All the proteins predicted to contain Pfam06766 and Pfam01185 were retrieved, (3) All protein sequences bearing the hydrophobin type I (C-X5–7-C-C-X19–39-C-X8–23-C-X5-C-C-X6–18-C-X2–13) or type II (C-X9-CC-X11-C-X14–16-C-X8-C-C-X10-C-X6–7) signature sequences were retrieved [−5) against each genome. A total of 243 hydrophobins were obtained. The classification was checked by Pfam carefully. These ascomycetes species were subdivided as entomopathogenic, mycoparasite, nematode parasitic, plant pathogenic and saprotrophic lifestyles. The data were analyzed by one-way analysis of variance (ANOVA). Significant differences were determined by Duncan’s multiple range tests. Data analyses were completed with SPSS 19.0  and GraphPad Prism 8.Hydrophobin-encoding genes were searched from the open genome of 47 Ascomycetes species following a protocol by Rineau et al. . Brieflyetrieved . Then, aCordyceps sensu lato species including Cordycipitaceae, Ophiocordycipitaceae and Clavicipitaceae and, in total, 90 amino acid sequences were downloaded from GenBank . The mummified pupae covered with mycelia before stroma development were designated as the sclerotium (ST). The mycelium knot tissue before stroma development was named as young primordium (YPR). The samples of stroma with lengths <1 cm, 1–2 cm and 5 cm were designated as the primordium (PR), young fruiting body (YF) and developed fruiting body (DF), respectively. The fruiting body with mature perithecia and ascospores was designated as the mature fruiting body (MF).The s report . SamplesWhen the fruiting body was cultivated on wheat medium, the hyphae covering the medium, which were cultured in the dark, were designated as hyphae (HY). Then, the hyphae were exposed under light irradiation. After being cultured under 12 h dark:12 h light for 4 days, the hyphae turned orange and were designated as colored hyphae (CH). The samples of YPR, PR, YF, DF, and MF were similar with those cultivated on insects.rpb1 gene (CCM_05485) was used as an internal standard [−ΔΔCt method was used to calculate the relative expression levels [https://matrix2png.msl.ubc.ca/) .RNA extraction and RT-qPCR assay were performed following the description before . The pristandard . The 2−Δn levels . The obtΔCmwc-1 [ΔCmvvd [Wild type, ΔCmwc-1  and ΔCmv [ΔCmvvd  strains C. militaris. Each hydrophobin gene had a different transcript pattern during the life cycle and different responses to light irradiation. The ascomycete fungi with a pathogenic lifestyle tended to be more favored by higher numbers of hydrophobin-encoding genes than the saprotrophic fungi. There was more variability among the class II members than class I, and only a few paralogous proteins were evolved by duplication. Multi-domain hydrophobins were only distributed in the species of Cordycipitaceae and Clavicipitaceae, whose function remains to be clarified.A total of four hydrophobin-encoding genes were identified and characterized in


Tested measures were investigated in separate trials and included (I) an increased amount of litter in the pen, (II) the reduction of stocking density to 25 kg/m2, and (III) the use of an alternative broiler breed (Rowan x Ranger). One-fifth of ESBL- and pAmpC- negative broilers (n = 18) per group were orally co-inoculated with two E. coli strains on the third day of the trial (seeder). One CTX-M-15-positive E. coli strain (ST410) and one CMY-2 and mcr-1-positive E. coli strain (ST10) were simultaneously administered in a dosage of 102 cfu. Colonization of all seeders and 28 non-inoculated broilers (sentinel) was assessed via cloacal swabs during the trials and a final necropsy at a target weight of two kilograms , d 47 (III)). None of the applied intervention measures reduced the colonization of the broilers with both the ESBL- and the pAmpC- producing E. coli strains. A strain-dependent reduction of colonization for the ESBL- producing E. coli strain of ST410 by 2 log units was apparent by the reduction of stocking density to 25 kg/m2. Consequently, the tested management measures had a negligible effect on the ESBL- and pAmpC- colonization of broilers. Therefore, intervention strategies should focus on the prevention of ESBL- and pAmpC- colonization, rather than an attempt to reduce an already existing colonization.The colonization of broilers with extended-spectrum β-lactamase- (ESBL-) and plasmid-mediated AmpC β-lactamase- (pAmpC-) producing Enterobacteriaceae has been extensively studied. However, only limited data on intervention strategies to reduce the colonization throughout the fattening period are available. To investigate practically relevant management measures for their potential to reduce colonization, a recently published seeder-bird colonization model was used. Groups of 90 broilers (breed Ross 308) were housed in pens under conventional conditions (stocking of 39 kg/m Specialobactams .Escherichia coli (E. coli) in the broiler production chain can partly be attributed to the very low colonization dosage. Recent findings have shown that 101 to 102 colony forming units (cfu) of orally administered ESBL- and pAmpC- producing E. coli can already lead to colonization of broiler chickens [E. coli from one flock to the subsequent flock in broiler fattening farms [Broiler chickens are considered as a reservoir for ESBL- and pAmpC- producing bacteria, it with frequently confirmed high prevalence , 4. Theschickens , 13. Cleng farms . Neverthng farms .2), and water and feed ad libitum. Due to the variety of study designs, no clear statement on the impact of practically relevant management measures on the colonization of broiler chickens with ESBL- and pAmpC- producing Enterobacteriaceae is possible. We aimed to investigate different management measures which are taken during the fattening process on their potential to reduce the spread of ESBL- and pAmpC- producing bacteria. We investigated the replacement of the broiler breed Ross 308 with the broiler breed Rowan x Ranger, increase of the common litter quantity of one kg/m2 to three kg/m2, and reduction of the common stocking density of 39 kg/m2 to 25 kg/m2. In our study, the investigated management measures had a negligible impact on the ESBL- and pAmpC- colonization of the broiler chickens.Different measures to influence the microbial composition of the broiler chickens’ guts and to affect the colonization of broiler chickens with ESBL- and pAmpC- producing Enterobacteriaceae are discussed in literature. One approach is the direct modulation of the broiler chickens’ microbiota to influence the early colonization with ESBL- and pAmpC- producing bacteria. For example, probiotics such as commercial Competitive Exclusion cultures and phytobiotics showed promising results to reduce the ESBL- and pAmpC- colonization of broiler chickens –18. AnotThis study was carried out following the National Animal Protection Guidelines. The protocol was approved by the German Animal Ethics Committee for the protection of animals of the Regional Office for Health and Social Affairs Berlin . All applicable national and institutional guidelines of the Freie Universität Berlin for the care and use of animals were followed. Experimental treatments of animals were classified as to lead to no worse than minor discomfort in the animals due to low pain of very short duration and were approved by LAGeSo.All trials were set up successively in the experimental facilities of the Center for Infection Medicine of the Department for Veterinary Medicine, Freie Universität Berlin, using controlled rooms with respective ventilation and HEPA filtration of the exhaust air. Before each trial, related room was disinfected using hydrogen peroxide fumigation, and the absence of ESBL- and pAmpC- producing bacteria was confirmed. Before entering the room, daily clothes and shoes were changed with trial specific clothing in an attached separate anteroom.2 cfu of one ESBL- and one pAmpC- producing E. coli on the third day of the trial , no environmental enrichment, and conventional feed and water ad libitum. The light regime was set to 11 hours of light and 13 hours of dark with a dimming period of 30 minutes. The floor temperature was decreased from 28°C (d 1–6), over 26°C (d 7–13) and 24°C (d 14–20) to 22°C  with a relative humidity of 55%. The broiler chickens were fed a commercial starter feed (d 1–7), grower feed, and a finisher feed  from a commercial broiler producer. Except for the finisher feed, coccidiostats were included in the feed (decoquinate and narasin/nicarbazin). Neither in the control nor the intervention groups antimicrobial agents were administered.In accordance with our recently established seeder-bird colonization model , a contrhe trial . Said coE. coli,The intervention groups I-III,  were kep2 was tripled from one kg/m2 to three kg/m2 over the course of the trial. We procured the litter which consisted of pelletized straw granules from a commercial broiler producer. The broiler breed , the number of broiler chickens (n = 90), the stocking density of 39 kg/m2, and the trial period of 36 days remained unchanged.the litter quantity in kg/m2 in Germany [2, as described in the Commission Regulation (EC) No. 543/2008 [2 to 7.2 m2, whilst the broiler breed , the number of broiler chickens (n = 90), the litter quantity of one kg/m2 and the trial period of 36 days remained unchanged.the common stocking density of 39 kg/m Germany  was redu543/2008 . For thi2, and the litter quantity of one kg/m2 remained unchanged.the broiler breed Rowan x Ranger (mixed gender) was included. In contrast to Ross 308, this breed is included in ‘slow-growing’ broiler concepts with an extended fattening period. The duration of the trial was set to 47 days so that the broilers reach the target weight of two kg. The number of broiler chickens per trial (n = 90), the stocking density of 39 kg/m® K 50 Gold Line containing 2.37% hydrogen peroxide and 0.015% peracetic acid . Following the second disinfection, the eggs for each experimental group were hatched in-house for 21 days. The eggs were incubated from day 1 to day 18 at 37.8°C and 60% relative humidity in a setter. From day 18 to day 21, the eggs were incubated at 37.2°C and 80% relative humidity for hatching.For each trial, we procured eggs from a commercial hatchery. The first disinfection using formaldehyde gas was already performed in the hatchery. After the transportation to the experimental facilities, the second liquid disinfection of the eggs was performed using WESSOCLEANBefore the trial, the absence of ESBL- and pAmpC- producing bacteria was confirmed in the eggshells and in the one-day-old broiler chickens as well as in the experimental room and on the equipment as described in our seeder-bird colonization model .E. coli strains to co-colonize our broiler chickens. One ESBL- producing E. coli strain  410; phylogenetic group B1; internal number 10716) published as R56 by Falgenhauer et al. [E. coli strain . Both strains were isolated in a previous research project [E. coli strains colonized the digestive tracts of broilers with high bacterial counts and without causing any clinical signs.We used two avian r et al.  and one  project  and were project . In thisE. coli strains was prepared, according to Robé et al. [The bacterial suspension for the inoculation of the broiler chickens with the ESBL- and pAmpC- é et al. .2 cfu E. coli of both bacterial strains in equal parts  were orally co-inoculated with 10al parts . These 1E. coli colonies. To confirm the ESBL-/ pAmpC- absence in the experimental room and the newly hatched broiler chickens, the agar was supplemented with two μg/ml cefotaxime . In order to process all other samples, a set of four chromogenic agar plates which has proven suitable for our study was used. The total count of E. coli colonies was determined using an agar plate without selective media (positive control). For the detection of the ESBL- E. coli 10716, one plate was supplemented with two μg/ml cefotaxime and four μg/ml enrofloxacin . For the detection of the pAmpC- E. coli 10717, one agar plate was supplemented with two μg/ml cefotaxime and seven μg/ml colistin . The fourth agar plate contained all three antibiotics in the given concentrations (negative control). All samples were incubated for 24 h at 37°C. Every untypical E. coli colony morphology was further analyzed using MALDI- TOF .The sample processing was identical to the one which was described in our seeder-bird colonization model . ChromogE. coli strains was monitored over the entire period of each trial via cloacal swabs. The investigated birds were randomly selected before the trial and repeatedly sampled during the trial, first of which was done 24 h post-inoculation (p.i.). Groups using the breed Ross 308 , and reduced stocking density (II)) were sampled for ten times during the 36 days of trial. The group using the breed Rowan x Ranger (III) was sampled 13 times during the 47 days of trial . To evaA necropsy was performed at a target weight of two kilograms for all sampled seeder- and sentinel-birds  and d 47 (III); Statistical analysis was carried out using SPSS Statistics 25 . Confidence intervals of proportions were calculated using the Clopper-Pearson method. For all analyses, the probability level to denote significance was set to 0.05.E. coli during the trials were analyzed via a logistic mixed regression model. The variables ‘group’, ‘sampling time’, and ‘animal’ were included in the analysis for both E. coli strains. To account for the repeated measurement of the same individual, ‘ID number’ and ‘sampling time’ were included in the analysis as random effects. Using backward selection, the best fitting model from a full model, including 2- and 3-way interactions, was obtained. The model with the lowest AIC value and lowest number of included effects was chosen, with an AIC change of two or less considered as equal. It resulted in the model with all main factors and the interaction term ‘animal and sampling time’. Residuals were inspected for normality and homoscedasticity for each sampling time point.The effects of the tested management measures on the colonization of broiler chickens with ESBL- and pAmpC- producing Mann-Whitney-U-Tests were performed to compare the level of colonization between the seeder- and sentinel-birds at necropsy. Kruskal-Wallis-Tests were performed to compare the final colonization of the digestive tracts of the tested groups to the control group at necropsy. Due to multiple comparisons in the Kruskal-Wallis-Test, the level of significance was set to 0.0167 (Bonferroni correction).E. coli strains was monitored via cloacal swabs throughout each trial, starting 24 h p.i. A strain-dependent course of colonization was evident in the investigated groups  of 0.18 (95% CI: 0.12–0.28). An increased amount of litter in the pen led to a higher risk of colonization for both E. coli strains (ESBL- E. coli HR 2.38 (95% CI: 1.49–3.82); pAmpC- E. coli HR 4.58 (95% CI: 2.09–10.06)), while the use of an alternative breed showed no effect on the hazard of colonization of broiler chickens with both E. coli strains (ESBL- E. coli HR 1.01 (95% CI: 0.66–1.56); pAmpC- E. coli HR 1.25 (95% CI: 0.65–2.40)).Colonization of broiler chickens with the ESBL- and pAmpC- d groups . ‘Group’d groups . CompareE. coli, the hazard rate of colonization was lower for sentinel-birds compared to seeder-birds with a hazard ratio of 0.11 (95% CI: 0.05–0.23). A reduced hazard for sentinel-birds could not be shown for the pAmpC- E. coli, while the interaction between ‘animal and sampling time’ was highly significant .At necropsy, respective contents of crop, jejunum, cecum, and colon were analyzed. As there was no significant difference in the level of colonization between the seeder- and sentinel-birds in any of the experimental groups  colonization with the resistant bacteria [2 [2 [2) was used in our related experimental group. A study by Persoons et al. [E. coli in broilers, with a higher risk for straw compared to wood curls. An effect of the bedding material on the ESBL- and pAmpC- colonization cannot be ruled out in our study, as all trials were conducted using pelletized straw granule. The fine litter structure of straw granule could cause a higher oral intake of the bedding material compared to other materials and a higher oral intake could have led to a higher (re-) colonization of the broiler chickens in our trial. As we want to mimic real farming conditions, we used pelletized straw granule which is most frequently used as bedding material in broiler fattening farms in Germany. As fresh litter is known to carry enteral bacteria [A similar approach of less contact to contaminated feces was taken for the experimental group with increased amount of litter in the pen. We hypothesized that the fecal droppings which harbor the resistant bacterial strains mix in the bedding material, for example, due to the movement of the chickens. Conversely, the cecum colonization of both the ESBL- and the pAmpC- bacteria . The desteria [2 , which ria [2 [2  in some s et al.  showed abacteria , the absbacteria .E. coli was apparent. Thus, our results support the findings of Richards et al. [An impact of the broiler breed on the microbial composition and colonization of the gut with resistant or pathogen bacteria is discussed , 35–37. s et al.  even thop.i. and colonization of all sentinel-birds 72 h p.i. [p.i. The lower prevalence may be due to the differences in the group size, with 20 broiler chickens in the colonization model compared to 90 broiler chickens in the current study. A bigger group size results in a larger pen, which might have affected the group dynamics. Compared to the control group, the prevalence in the intervention groups might be due to an impact of the applied intervention measure itself or can partly be a result of the variability of cloacal swab samples. As fermentation of the bacteria takes place in the ceca [Various scenarios can lead to ESBL- and pAmpC- colonization of broiler chickens, as reviewed by Dame-Korevaar et al. . Transmi2 h p.i. . In contthe ceca , and thethe ceca , the detthe ceca  pointed the ceca . Neverththe ceca .2, and (III) the use of an alternative broiler breed, are negligible on the ESBL- and pAmpC- colonization of broiler chickens. Nevertheless, these results do not entirely exclude the possibility of management measures reducing the colonization of broiler chickens with ESBL- and pAmpC- producing bacteria. Particularly, microbiome-directed measures could represent a promising effect as they directly address the digestive tract. Probiotics such as commercial Competitive Exclusion cultures and phytobiotics already showed positive results of reducing the ESBL- and pAmpC- colonization of broiler chickens [To summarize, the effects of the tested management measures, namely (I) an increased amount of litter in the pen, (II) the reduction of stocking density to 25 kg/mchickens –18 and sS1 Fignd week of trial, 6–7 = 3rd week of trial, 8–9 = 4th week of trial, 10 = 5th week of trial; Error bar = 95% Confidence interval.Samplings: 1 = 24 h post-inoculation, 2 = 72 h post-inoculation, 3–5 = 2(TIF)Click here for additional data file.S2 Figp < 0.001 .*** (TIF)Click here for additional data file.S1 TableE. coli, 10717 = pAmpC- E. coli; See = seeder-birds, Sen = sentinel-birds; sampling: 1 = 24 h post-inoculation, 2 = 72 h post-inoculation, 3–5 = 2nd week of trial, 6–7 = 3rd week of trial, 8–9 = 4th week of trial, 10–11 = 5th week of trial, 12–13 = 6th week of trial.* Prevalence in percent (%); 10716 = ESBL- (DOCX)Click here for additional data file.S2 TableE. coli, 10717 = pAmpC- E. coli; all data shown are log10 transformed (log10 cfu/g); ± CI = ± 95% Confidence interval.10716 = ESBL- (DOCX)Click here for additional data file.


Haemophilus influenzae, which is one of the leading causes of community-acquired bacterial pneumonia (CABP). The evaluation of antimicrobial agents against H. influenzae using standard murine infection models is challenging due to the low pathogenicity of this species in mice. Therefore, 24-h dose-ranging studies using a one-compartment in vitro infection model were undertaken with the goal of characterizing the magnitude of the ratio of the area under the concentration-time curve (AUC) to the MIC (AUC/MIC ratio) associated with efficacy for a panel of five H. influenzae isolates.Omadacycline is a novel aminomethylcycline with activity against Gram-positive and -negative organisms, including  Haemophilus influenzae, which is one of the leading causes of community-acquired bacterial pneumonia (CABP). The evaluation of antimicrobial agents against H. influenzae using standard murine infection models is challenging due to the low pathogenicity of this species in mice. Therefore, 24-h dose-ranging studies using a one-compartment in vitro infection model were undertaken with the goal of characterizing the magnitude of the ratio of the area under the concentration-time curve (AUC) to the MIC (AUC/MIC ratio) associated with efficacy for a panel of five H. influenzae isolates. These five isolates, for which MIC values were 1 or 2 mg/liter, were exposed to omadacycline total-drug epithelial lining fluid (ELF) concentration-time profiles based on those observed in healthy volunteers following intravenous omadacycline administration. Relationships between change in log10 CFU/ml from baseline at 24 h and the total-drug ELF AUC/MIC ratios for each isolate and for the isolates pooled were evaluated using Hill-type models and nonlinear least-squares regression. As evidenced by the high coefficients of determination (r2) of 0.88 to 0.98, total-drug ELF AUC/MIC ratio described the data well for each isolate and the isolates pooled. The median total-drug ELF AUC/MIC ratios associated with net bacterial stasis and 1- and 2-log10 CFU/ml reductions from baseline at 24 h were 6.91, 8.91, and 11.1, respectively. These data were useful to support the omadacycline dosing regimens selected for the treatment of patients with CABP, as well as susceptibility breakpoints for H. influenzae.Omadacycline is a novel aminomethylcycline with activity against Gram-positive and -negative organisms, including  Streptococcus pneumoniae, Haemophilus influenzae is a leading cause of CABP  is a major cause of morbidity and mortality worldwide –3. After– of CABP , 4–6. Rato 43.0% , while 3to 43.0% , 10.in vitro activity against the most common Gram-positive and -negative pathogens associated with CABP, including H. influenzae (in vivo studies were carried out to characterize the pharmacokinetics-pharmacodynamics (PK-PD) of omadacycline against Staphylococcus aureus and S. pneumoniae  is generally considered to be the PK-PD index most closely associated with efficacy for tetracyclines  examined.The inocula evaluated in the frequency of resistance studies failed to produce or identify a drug-resistant subpopulation for all isolates evaluated, as presented in in vitro infection model, as evidenced by the agreement between observed and targeted concentrations shown for all data evaluated in The targeted concentration-time profiles representing the beta and terminal half-lives of omadacycline in human epithelial lining fluid (ELF) were well simulated in the one-compartment r2) value of 0.964 and a slope of 0.970 (which is close to 1) for the observed versus model-predicted concentrations . No bias was evident when goodness-of-fit plots were examined (data not shown). Using the final PK model, omadacycline total-drug ELF AUC values were computed through numerical integration for each dosing regimen studied in the one-compartment in vitro infection models, which are described in Pharmacokinetic (PK) models were fit to the samples collected for the evaluation of the omadacycline concentration-time profiles. The final PK model, which was a one-compartment model utilizing two different clearance terms over the 24-h study duration, fit the data with excellent precision, as evidenced by a coefficient of determination , intermediate exposures reaching only net bacterial stasis at 24 h, and higher exposures achieving reductions in the bacterial burden over the 24-h period.Across the omadacycline dosing regimens evaluated in the one-compartmen10 CFU/ml from baseline at 24 h and omadacycline total-drug ELF AUC/MIC ratio were evaluated based on the data for each individual H. influenzae isolate as well as a pooled collection. As evidenced by a high r2 of 0.88 and the dispersion of data about the fitted function, the relationship between the change in the log10 CFU/ml from baseline at 24 h and total-drug ELF AUC/MIC ratio shown in H. influenzae isolates with MIC values ranging from 1 to 2 mg/liter. As shown in 10 CFU/ml from baseline at 24 h in the absence of drug (E0) was 2.80 (0.34), the maximum change in the log10 CFU/ml from E0 (Emax) was 8.81 (0.75), the Hill coefficient was 1.66 (0.29), and the omadacycline total-drug ELF AUC/MIC ratio associated with half-maximal effect (50% effective concentration [EC50]) was 9.29 (1.13). The magnitude of the total-drug ELF AUC/MIC ratio associated with net bacterial stasis and 1- and 2-log10 CFU/ml reductions from baseline at 24 h based on the pooled data were 5.87, 7.87, and 10.4, respectively.The relationships between the change in the log10 CFU/ml from baseline at 24 h and the total-drug ELF AUC/MIC ratio for each H. influenzae isolate also described the data well . Parameter estimates (standard errors) for these Hill-type models are shown in 10 CFU/ml reductions from baseline at 24 h were 6.91 , 8.91 , and 11.1 , respectively.Hill-type models describing the relationship between the change in the logH. influenzae isolates to omadacycline. The second objective was to determine the magnitude of the omadacycline AUC/MIC ratio associated with efficacy for the above-described panel of H. influenzae isolates evaluated using a one-compartment in vitro infection model.The studies described herein were carried out to accomplish two objectives. The first was to evaluate the frequency of mutation of clinical H. influenzae isolates evaluated could not be identified for bacterial populations less than or equal to 2.9 × 109 CFU/ml at omadacycline concentrations representing 3 or 5 times the baseline MIC. These values are similar to those identified by Clark et al. . The median omadacycline total-drug ELF AUC/MIC ratio associated with net bacterial stasis and the 1- and 2-log10 CFU/ml reductions from baseline at 24 h for the H. influenzae isolates studied were 6.91, 8.91, and 11.1, respectively. As described above, PK-PD targets for H. influenzae were characterized using an in vitro infection model, given the difficulty of achieving sufficient growth and potential for auto-clearance of the pathogen in in vivo infection models. Thus, while information derived from in vitro infection models represents an optimal approach to characterize the PK-PD targets for H. influenzae efficacy, such targets may be overestimated relative to what is expected using an in vivo infection model as a result of the enhanced effect of growth media on bacterial growth and the lack of an immune system.Results obtained from the dose-ranging studies demonstrated that the data for the five isolates comodeled well. As evidenced by a high in vitro model than would have been possible using a standard murine infection model.The evaluation of effect site exposures, which for CABP is total-drug ELF, and thus the characterization of total-drug ELF AUC/MIC ratio targets represented an important strength of the studies described herein. Mimicking the shape of total-drug ELF, which involved applying two different half-live estimates observed in healthy volunteers , was easin vitro susceptibility testing for the antimicrobial agent against a given pathogen  and JMI Laboratories . The challenge panel included four clinical isolates and one ATCC reference strain (H. influenzae ATCC 49247). Omadacycline was provided by Paratek Pharmaceuticals, Inc. .A panel of five Haemophilus test medium (HTM), in triplicate, over a 2-day period and are reported as the modal values.Omadacycline MIC values were determined for the challenge isolate panel according to Clinical and Laboratory Standards Institute guidelines  using ag9 CFU/ml, was plated using 200-μl volumes on 10- by 100-mm HTM agar plates supplemented with three times or five times the omadacycline baseline agar dilution MIC. The bacterial concentration in the suspension was determined by quantitative culture using drug-free agar. The inoculated agar plates were incubated at 35°C and then observed for growth, with individual colonies counted 48 h postincubation. The frequency of resistance was determined as the ratio of the number of colonies found growing on the drug-supplemented agar plates to the total number of colonies plated. If no isolates were observed after 48 h of incubation, then the resultant mutation frequency was determined to be less than that of the inoculum evaluated.The frequency of omadacycline resistance was estimated for each challenge isolate. In brief, 2 ml of log-phase growth suspension, consisting of approximately 1.0 × 10in vitro infection model, as previously described , the challenge organism, and magnetized stir bars to ensure that the test compound, media, and organism remained in a homogenous state. The central infection compartment was placed on a magnetic stir plate, which was then housed in a temperature-controlled incubator set to 35°C. The bacterial suspension in the central compartment was then exposed to dynamic concentration-time profiles of omadacycline designed to simulate human ELF concentration-time profiles in healthy volunteers following i.v. administration . Isolates were collected from the overnight cultures, suspended in HTM broth, and grown to mid-logarithmic growth phase in an Erlenmeyer flask immersed in a shaking water bath set to 35°C and 125 rotations per minute. The optical density of the bacterial suspension growing in the flask was compared to those of previously completed growth curves using a spectrophotometer set to a wavelength of 630 nm. Isolates were then inoculated in the system to achieve an initial bacterial burden of 1.0 × 10drug ELF . Omadacyin vitro system, a second 1-ml sample aliquot was collected at 1, 2, 3, 5, 7, 11, 13, and 23 h postinoculation. All samples for evaluation of omadacycline concentration were immediately frozen at –80°C until they were assayed via liquid chromatography-tandem mass spectrometry (LC/MS/MS).In order to evaluate the effect that omadacycline had on the bacterial suspension in the central compartment, a series of 1-ml samples were collected for the determination of bacterial density at 0, 2, 4, 8, 12, and 24 h. In order to eliminate the potential for drug carryover, each bacterial sample was centrifuged, aspirated, and resuspended to the initial sample volume with sterile normal saline in duplicate to eliminate drug carryover. The washed sample was diluted serially by 10-fold volumes in sterile normal saline, cultured on chocolate agar, and placed in a humidified incubator for 24 h. In order to measure drug concentrations in the r2) value ranging from 0.9926 to 0.9992. The lower limit of quantification was 0.100 mg/liter.Calibration standard and quality control samples were prepared in HTM and processed concurrently with collected samples. All samples were assayed via LC/MS/MS on a Sciex 5500 mass spectrometer using a Thermo Hypercil Gold C8 column (3 by 100 mm) with a mobile phase of 10 mM ammonium formate (pH 3.0) and acetonitrile. The standard curve for omadacycline was linear, ranging from 0.10 to 10.0 mg/liter, with a correlation coefficient (in vitro infection model. Given the assumption that protein binding in ELF is negligible, total-drug ELF AUC values were evaluated and not further adjusted to account for protein binding. Data from the omadacycline dose-ranging studies were evaluated using Hill-type models and nonlinear least-squares regression. All data were weighted using the inverse of the estimated measurement variance. The relationship between the change in log10 CFU/ml from baseline at 24 h and total-drug ELF AUC/MIC ratio was evaluated by utilizing the broth dilution MIC values. The magnitude of the total-drug AUC/MIC ratio associated with net bacterial stasis and 1- and 2-log10 CFU/ml reductions from baseline at 24 h was determined for each H. influenzae isolate individually and based on the pooled data for the isolates studied.PK models were fit to the samples collected for the evaluation of the total-drug ELF concentration-time profiles. Omadacycline total-drug ELF AUC values were calculated for each dosing regimen simulated in the one-compartment